Cre recombinase forms a tetrameric complex that splices DNA molecules containing the 34-bp recombination target (RT) site loxP (Sternberg and Hamilton, 1981, J Mol Biol, 150, 467-86), recombining two DNA molecules in trans to accomplish an insertion or translocation event, or in cis to achieve either gene excision or inversion, depending on the relative orientation of the loxP sites. Cre recombinase has been used to generate conditional gene knockouts, where a gene of interest is flanked by loxP sites (‘floxed’) (Gu et al., 1993, Cell, 73, 1155-64). Expression of Cre recombinase under the control of promoters that are specific for particular tissues or developmental stages abrogates gene function by physical excision from the genome. The utility of this system depends on the functional autonomy of Cre recombinase: the enzyme requires no other factors to splice DNA, and is capable of modifying genomes in non-replicating cells, where the efficacy of gene conversion via double-strand break (DSB) induced homologous recombination is expected to be low (Saleh-Gohari and Helleday, 2004, Nucleic Acids Res, 32, 3683-8; Rothkamm et al., 2003, Mol Cell Biol, 23, 5706-15).
Another application for Cre recombinase is recombination-mediated cassette exchange (RMCE) (Bouhassira et al., 1997, Blood, 90, 3332-3344), also known as double-reciprocal crossover (Schlake and Bode, 1994, Biochemistry, 33, 12746-12751; Seibler and Bode, 1997, Biochemistry, 36, 1740-1747) or double-lox replacement (Bethke and Sauer, 1997, Nucleic Acids Res, 25, 2828-34; Soukharev et al., 1999, Nucleic Acids Res, 27, e21). In this approach, (reviewed in Turan et al., 2013, Gene, 515, 1-27) recombination between DNA molecules that share two neighboring heterologous RT sites accomplishes the exchange of the bounded genetic interval (the cassette) between the sites. This has been demonstrated using both Flp and Cre recombinase with heterologous RT variants (Bethke and Sauer, 1997, Nucleic Acids Res, 25, 2828-34; Bouhassira et al., 1997, Blood, 90, 3332-3344), as well as simultaneously with Cre and the Flp recombinases (Anderson et al., 2012, Nucleic Acids Res, 40, e62). Although RMCE has so far only been demonstrated with wild-type recombinase proteins and RT sites, the approach has many attractive features as a tool for genome engineering. First, it has a higher efficiency for gene conversion than does Cre-mediated insertion, as it does not require survival of insertional events that are susceptible to reversal by excision (Bethke and Sauer, 1997, Nucleic Acids Res, 25, 2828-34). Second, the cassettes that are exchanged are precisely demarcated, yielding truly ‘scarless’ genomic surgery. Third, the process requires less Cre protein than recombinational insertion, resulting in less cytotoxicity (Bethke and Sauer, 1997, Nucleic Acids Res, 25, 2828=34). Finally, the autonomy of Cre as a recombinase suggests that RMCE could prove to be effective in terminally differentiated cells, in contrast to strategies for gene conversion that rely upon homology directed repair.
One impediment to broader use of Cre recombinase is the inflexibility of the binding site specificity. In contrast to DNA binding proteins whose specificity derives from the assembly of small recognition modules such as zinc finger or TAL effector domains, Cre recombinase interacts with DNA through large interfaces that defy a modular decomposition. Accordingly, broader application of the Cre recombinase system is limited by the fixed sequence preferences of Cre, which are determined by both the direct DNA contacts and the homotetrameric arrangement of the Cre monomers. Thus, there is a need in the art for a method to break the symmetry of Cre recombinase such that its use may expanded to broader applications. As such, there is an unmet need for recombination systems that are not limited to wild type recognition sites and moreover that are not restricted to palindromic symmetry of recognition sites, thus enabling recombination of any desired recombination site.